XENOTRANSPLAMATION II
`
`Abstract# 845
`Poster Board #-Session: P232-I1
`ROLE OF NITRIC OXIDE AND ENDOTHELIN IN ISCHEMIA/
`REPERFUSION INJURY IN THE RAT PANCREAS. NicholasT. Stowe,l
`AM V. Robinson,l Mike S. Simonson? James A. Schulak.'.' 'Surgery, Case
`Western Reserve University, Cleveland, OH; 21nternal Medicine, Case
`Western Reserve University. Cleveland, OH: *Surgery. University Hospitals,
`Cleveland, OH.
`Both nitnc oxide (NO) and endothelin (ET-I) control local vascular resistance and
`have been implicated in ischemidreperfusion (I/R) injury in various organs. The
`purpose of this study was to assess the role of NO and ET-I following I/R injury
`in the pancreas by infusing the NO precursor I-arginine. In anesthetized Sprague
`Dawley rats, the pancreas was isolated through a midline incision. Pancreatic blood
`flow (PBF) was measured in the inferior splenic artery with an ultrasonic flow
`probe before and 90 min after ischemia. Two groups were studied. In the control
`ischemia group, the pancreas was subjected only to 60 min of ischemia by occluding
`the inferior splenic artery. In the treated ischemia group, the NO precursor, 1-
`arginine (4Oomghg. i.v.). was infused for 30 min pnor to ischemia. At the end of the
`experiments, blood samples were taken for amylase and lipase activity. In selected
`experiments, plasma ET-I levels and immunohistochemical analysis of ET-I were
`done. In
`the untreated
`ischemia group (n=4).
`ischemia caused a
`7X.lfl.9%(meamtSEM) decrease in PBF from 0.39M.04 mVmin to 0.07M.01 ml/
`min. In contrast, rats pretreated with I-arginine (n=3) experienced only a 41 .%10.6%
`drop in PBF from 0.76M.02 ml/min to 0.46M.05 ml/min, a value significantly
`different (p4.05) from the untreated controls. Lipase values were significantly
`lower after ischemia in the I-arginine treated group at 34.5f11.5 U/L (p<O.05)
`compared to the untreated ischemic group. Amylase values were also lower at
`2688f530 U/L (p<O.lO). In a separate group of experiments ( n d ) . plasma ET-I
`levels were significantly elevated following 60 min of ischemia (1.17M.12 vs
`0.66M.15 pg/ml, in controls, p<O 05). lmmunohistochemical localiiation of ET-I
`peptide following ischemia exhibited increased staining in the vascular endothelium.
`I/R injury in the pancreas is characterized by a decrease in PBF, an increase in
`amylase and lipase activity and an increase in plasma ET-I levels. Pretreatment
`with I-arginine significantly ameliorated the drop in PBF and decreased the levels of
`lipase and amylaw following ischemia. Use of the NO precursor. I-arginine. can
`enhance the production of NO which could antagonize the vasoconstrictor effects
`of ET-I. Such an approach may be useful in the transplantation setting to minimize
`I/R injury and preserve graft function.
`
`POSTER SESSION 11:
`I1
`XENOTRANSPLANTATION
`
`Abstract# 846
`Poster Board #-Session: P233-I1
`LYTRAGRAFl' EXPRESSION OF PROTECllON GEhES HO-1 Ah'D Bd-
`2 IN PRIiMARY HEART XENOGRAFTS: A LACK OF PROTECTION
`EF%E4TACAINSTXENOGRAFTHYPERACWI'EREJE(JT1ONNUCED
`BY HYPER-IMMUNE SERA. Gordon D. Wu. Yang-Sung Jin. Vaughn A.
`Starnes, Donald V. Cramer. 'Cardiothoracic Surgery. Keck School of
`Medicine, USC. Los Angeles, CA.
`Heme-oxygenase-l (HO-1) and Bcl genes are expressed by long-surviving rodent
`xenografts and have been suggested to have a protective role in accommodating
`xenografts against antibody InJUQ!. Expression of these genes by organ xenografts in
`early stages of humoral rejection has not been carefully examined. We have recently
`conducted series of studies on the hamster-to-rat heart transplant model to see
`whether intragraft expression of protection genes is associated with protecting
`xenograft from antibody rejection. First, we \tudied intragraft expression of HO-1
`and Bcl-2 genes at different time points (0.60 minutes, 12,24,48,72 and 96 hours)
`post-transplantation (PTx). All the xenografl ramples (n=3 in each Group) were
`snap-frozen, cryostat sectioned and subjected to immunohistochemistry using
`antibodies against HO-I. Bcl-2, and Bax. We found that hamster heart tissue with
`cold preservation (Group TO) expressed strong HO-I protein. and heart xenografts
`in other groups exhibited moderate HO-I staining. lmmunostain for Bcl-2 was also
`positive in all of the xenografts except Group TO. On the other hand, immunostaining
`for Bax (apoptosis gene) were mild to moderately positive in the xenografts of
`Groups T12h (12 hours PTx), T24h (hours). T48h (hours) and 72h (hours) but
`negative at Groups TO and T60m (minutes), indicating the involvement of
`programmed cell death in xenograft during pnmary rejection. To evaluate whether
`HO-I and BcI expression can protect grafts from antibody injury, we conducted
`antiserum passive transfer experiments in which hamster heart xenografts were
`challenged with anti-hamster sera (0.5 ml 1.V ) at different time points PTx. We
`found that xenografts challenged with antisera at 60 minutes (n=5) and 12 hours
`(n=5) were hyperacutely rejected (10-18 minutes) despite of HO-I and Bcl-2 gene
`expression by the heart grafts. indicating that intragraft expression of these protection
`genes was not effective for protecting the xenograft from humoral injury. We conclude
`that HO-I and Bcl-2 gene expression in hamster hem xenografts in early stage of
`the humoral rejection has minimal protective effect on xenograft survival, 11 may
`simply reflect a stress-related cellular response of the xenograft tissue to various
`stimuli dunng xenograft rejection.
`
`Abstract# 847
`Poster Board #-Session: P234-I1
`EVALUATION OFDEFEREhToGGALGLYCOCONJUGATES FOR USE
`IN XENOTRANSPLANTATATION. Alexander Schwarz,' Ivona Bakaj,l
`Patrick Birch,' Joanna Fesi,' Anna Nepomich,' Cianna Cooper,' Margaret
`A. Velardo,' Aileen Stark,' Lisa E. Diamond,' John S. Logan,' Guerard W.
`Byme.[ 'Nextran, Princeton. NJ.
`Porcine organs, are rapidly rejected after transplantation into pnmate recipients
`due to the presence of pre-existing immunoglobulins that bind to terminal galactose
`al.3 galactose residues (a-Gal) present on porcine glycoproteins and glycolipids.
`These antibodies are responsible for initiating hyperacute rejection (HAR) of porcine
`organs, and under conditions that block HAR. appear to be induced and contribute
`to the process of acute vascular rejection. Currently available immunosuppressive
`reagents have been largely ineffective at controlling the induction of anti-Gal
`antibodies. Non-antigenic hapten polymers have been shown to be effective materials
`for controlling humoral immune responses in various model systems. As a
`preliminary step toward developing an a-Gal containing polymer to control the
`induced a-Gal antibody we have developed a senes of a-Gal glycoconjugates and
`tested their ability to block anti-Gal antibody binding in vitro. A galactose a1.3
`galactose p 1.4 GlcNAc trisaccharide (TRFA) with a 5 carbon spacer arm was
`coupled to a variety of polymeric backbones including dextran. branched
`polyethylene glycol (PEG). polymethacrylamide (PMA). and poly-L-lysine. The
`ability of these a-Gal conjugates to block anti-Gal IgG and IgM binding was
`determined relative to TRFA in an ELlSA assay using a defined HSA-Gal
`glycoconjugate substrate. The ability of these conjugates to block complement
`dependent cytotoxicity was also determined. When the TRFA sugar was coupled
`to a branched amino PEG carrier using I-ethyl-3(3-dimethylaminopropyl)
`carbodiimide ( E X ) . a uniform product with 8 a-Gal residues was obtained. This
`material was only marginally more effective than TRFA in blocking anti-Gal antibody
`interactions. In contrast a polymer of the branched 8 arm PEG and linear conjugates
`made using Dextran. poly-L-lysine or polymethacrylamide, all with similar antigen
`densities. were much more effective inhibitors of anti-Gal antibodies and anti-Gal
`dependent cytotoxicity. a-Gal conjugates similar to these may be useful both to
`block anti-Gal interactions In vivo and to Specifically control the induced anti-Gal
`immune response.
`
`Poster Board #-Session: P235-I1
`Abstract# 848
`HUMAN MONOCYTES PLAY AN IMPORTANT ROLE IN ISDIRECT
`ANTIGEN PRESENTATION AND COSTIMULATION TOT CELLS
`DURING HUMAN ANTI-PORCINE IMMUNE RESPONSES. He Xu,
`Douglas K. Tadaki, Patrick J. Blair, Francis Cruzata, David M. Harlan, Allan
`D. Kirk.
`Unlike peripheral blood mononuclear cells (PBMC), purified T cells proliferate
`poorly in response to pig endothelial cells (PEC) suggesting that monocytes may
`play a key role in xenogeneic T cell responses. We studied the role of monocytes in
`indirect antigen presentation and costimulation to T cells during human anti-pig
`immunoresponses. The CD40 and CD80 expression on monocytes from a human
`PBMC-PEC coculture was evaluated by FACS. Unlike resting monocytes expressing
`only CD86, monocytes from PBMC-PEC cocultures expressed CDBO and CD40
`as early as 24 hours and high level exprersion at 72 hours of coculture. This
`expresrion was dependent on the presence of T cells, as purified monocytes did not
`up-regulate CD40 and CDRO when incubated alone with PEC. Incubation of purified
`T cells with monocytes harverted from monocyte-PEC cocultures showed up-
`regulation of CD40 and CDRO on monocytes suggesting that these monocytes are
`activated in the presence of naive T cells and that PEC interact with monocytes to
`condition them for activation. Monocytes collcctcd from monocyte-PEC coculture
`werc incubated with purified autologous T cells in the presence or absence of anti-
`CDXO or anti-CDR6 monoclonal antibodies to study the role of conditioned
`monocytes on T cell proliferdtion and costimulation Unlike resting monocytes.
`PEC-activated monocytes induced T cell proliferation and this proliferation was
`inhibited by 8 7 blockade suggesting a key role for B7/CD28 interactions between
`PEC-activated monocytes and T cells i n xenograft rejection. In summary. human
`monocytes exposed to PEC are conditioned to up-regulate costimulatory molecules
`upon expose to T cells. PEC-conditioned monocytes induce T cell proliferation by
`indirect presentation. and costimulatory blockade against B7 inhibits T cell
`proliferation. Our findings suggest that monocytes play a key role in indirect
`antigen presentation and in providing costimulation to T cells through B7/CD28
`pathway. This interaction can occur distant from the initial rile of xenoantigen but
`monocytes remain void of costimulatory signals until interaction with T cells. This
`pathway suggests that human, not porcine. costimulatory molecules are critical
`during xenografts. and demonstrates a mechanism hy which monocyter traffic to
`secondary lymphoid tissue without providing costimulatory signals in the peripheral
`circulation
`
`

`

`Poster Board #-Session: P236-I1
`Abstract# 849
`DEVELOPMENT AND CHARACI'ERIZATION OF ANTI-GAL B-CELL
`RECEPTOR TRANSGENIC GAL-/- MICE. Hui Xu, Ymg Lei, Ajay Sharma,
`Hua Wan, Jeaninne Okabe, John S. Logan, Guerard W. Byme. 'Nextran Inc.
`Princeton, NJ.
`The successful clinical application of xenotransplantation using organs derived
`from pigs IS currently limited by the development of acute vascular rejection a
`process that is thought to involve an induced humoral immune response, largely
`directed towards the a-Gal antigen. Successful transplantation of pig organs into
`humans may require the development of methods for removal or neutralization of
`anti-Gal antibodies and anti-Gal producing B-cells. The large diversity of the B cell
`repertoire makes it difficult however to study anti-Gal B cells. To address this issue
`we have established a transgenic mouse model (BCR;Gal-/-) for investigating anti-
`Gal B cells by introducing a transgene encoding both heavy and light chains for an
`anti-Gal IgM antibody into Gal-/- mice. ELISA analysis of serum from BCR;Gal-/
`
`-. BCR;Gal+/- transgenic mice and Gal-/- mice demonstrates elevated expression of
`
`anti-Gal antibodies in BCR;Gal-I- mice compared to nontransgenic Gal-/- animals.
`Anti-Gal antibody expression in BCR;Gal-/- mice could be increased by
`immunization with an ovalbumin-Gal glycoconjugate in vivo and through stimulation
`with LPS in vitro indicating the presence of functional anti-Gal B cells. Multiparameter
`flow cytometric analysis indicates that 7040% of splenic and peritoneal B cells
`excluded endogenous immunoglobulin gene rearrangement and expressed the
`transgene. FACS analysis of the BCR;Gal-/- B cells identifies them as a population
`of CDS-CD23+ conventional B cells. The majority of these B cells expressed anti-
`Gal receptors on the surface, as identified by FITC-Gal-BSA staining. These
`observations suggest that this model can be used to establish a reliable source of
`anti-Gal B cells which potentially could be used to test the effectiveness of
`immunosuppressive reagents.
`
`Poster Board #-Session: P237-11
`Abstract# 850
`ANTI-GAL ANTIBODY GENE USAGE IN NAIVE AND POST
`XENOCRAFI'GAL-/- MICE. Hui Xu,' Ajay Sharma,' Ltblng Chen,' Caren
`Harrison,' Yuanyuan Wei,' Anita S.-F. Chong,z John S. Logan,' Guerard W.
`Byme.' 'Nextran Inc, Princeton. NJ.
`Naturally occurring antibodies that bind to terminal galactose al.3-galactose
`carbohydrate structures (Gal) are present in humans and Old World Monkeys but
`are negatively regulated in other mammalian species since they express Gal cpitopes
`on their cell wrfaces. A Gal knockout m o w (Gal-/-) model. generated by homologous
`disruption of the a1 ,3-galactosyltransferase gene, is capable of producing natural
`anti-Gal antibodies. To study the genetic control of the anti-Gal response, we have
`generated anti-Gal hybridomas from Gal-/- mice and analyzed the VH genes of
`these antibodies from nayve animals and from mice stimulated by rat heterotopic
`heart transplantation. Six IgM anti-Gal hybridomas derived from naive Gal-/- mice
`exhibited anti-Gal binding activity with some cross reactivity to related carbohydrate
`structures. These naive anti-Gal antibodies used five different VH genes in a germline
`configuration. Anti-Gal IgM hybridomas isolated after a rat heterotopic heart
`xenograft (4 and 21 days) utilized germline VH gene segments from the VH7183
`family and exhibited less cross reactivity. In contrast, we have predominantly
`isolated IgG anti-Gal hybridomas from mice 21-days after rat heterotopic heart
`xenografts, indicating an isotype switch. Nine of the IgG anti-Gal hybridomas
`secreted the lgG3 subclass and one produced IgGI. Sequence analysis of the VH
`gene usage from the induced anti-Gal IgG antibodies demonstrated a restncted gene
`utilization. VHJ606-14A. Our results demonstrate that the anti-Gal response in
`nai've Gal-/- mice i* encoded hy multiple germline progenitors. In response to a
`xenograft. the induced anti-Gal antibodies exhibited a restncted gene usage and
`somatic mutations. indicating a positive selection
`
`Poster Board #-Session: P238-I1
`Abstract# 851
`ANTI-GAL MONOCLONAL ANTIBODIES INDUCE PIG
`MICROVASCULAR E"IlLELIALCELL(PMVEOACIWATI0NAND
`APOPTOSIS. Hui Xu. Bashoo Naziruddin, Yuanyuan Wei, Libing Chen,
`John S. Logan, Guerard W. Byme. 'Nextran Inr. Princeton, NJ
`We have produced a series of anti-Gal hybndomas from Gal -/- mice after rat to
`mouse heterotopic cardiac xenografts. These monoclonal anti-Gal antibodies provide
`us an opportunity to study the effects that anti-Gal antibodies may have on
`endothelial cells in a very defined manner. All IgG3 anti-Gal monoclonal antibodies
`bound to pig red blood cells (RBC). and induced complement dependent lysis. In
`contrast. an lgGl anti-Gal antibody tailed to activate complement. Incubation of
`anti-Gal monoclonal antibodies with cultured porcine microvascular endothelial
`cells (PMVEC) for 2-4 hours resulted in activation of endothelial cells as determined
`by the induction of E-selectin (CD62E). This activation was anti-Gal specific since
`the induction of CD62E by anti-Gal hybridomas was blocked by IOmM free a-Gal
`trisacharride and since a mouse IgG isotype control failed to induce CD62E
`expression. When antibodies were incubated with endothelial cells overnight at hlgh
`concentration. anti-Gal antibodies also induced apoptosis as determined by Annexin
`V staining. The induction of apoptosis was dose dependent within the range of S-
`SO pg/ml and isotype controls. mouse lgC3 and IgGl. did not induce significant
`apoptosis. The induction of endothelial cell activation. or apoptosis showed no
`
`XENOTRANSPLANTATION II
`
`apparent relationship to the affinity of the anti-Gal antibody. These data suggest
`that induced anti-Gal antibodies are capable of effecting the endothelial cells of a
`graft and could induce the formation of a procoagulant vasculature, without the
`need of complement mediated damage.
`
`Poster Board #-Session: P239-I1
`Abstract# 852
`PIG-TO-PRIMATE RENAL XENOTRANSPLANTATION -
`PREDOMINANT GLOMERULAR INJURY IN LONG TERM
`FUNCTIONING H-CD55 TRANSGENIC KIDNEYS. Jan Schmidtko,'
`Martin Loss.' Ergin Kilic,? Ralf Roland Lorenz,' Jens Martin Hecker,' Richard
`Appiah,' Robert Kun2.l Juergen Klempnauer,' Udo Helmchen,2 Michael
`Winkler. I 'Klinik fur Viszeral- rind Transplantationschrriir~ie, Medizinisrhe
`Hochschule Hannover, Hannover. Germany; 'Institrct fiir Pathologie.
`Universitaets-Klinikum Eppendorf, Hamburg, Germany; 'Zentrum
`Anaesthesiologie, Medizinische Hochschule Hannover, Hannover.
`Germany.
`The introduction of transgenic pigs expressing human complement regulatory
`molecules such as h-CD5S or h-CDS9 into pig to primate discordant kidney
`xenotransplantation has lead to xenograft survival times consistently above 1 week.
`This allows for a more refined analysis of discordant xenograft pathology during
`longer graft survival times. Methods: H-CDSS positive porcine kidneys were
`transplanted in cynomolgus monkeys under postoperative cyclophosphamide,
`cyclosporine A and steroid baseline immunosuppression and CI -1nh treatment for
`episodes of acute vascular rejection (AVR). We here report on a subgroup of
`experiments ( n 4 ) with graft survival times of more than 2 weeks. In all experiments
`graft biopsies were obtained at regular intervals. Histological monitoring was
`performed by means of immunohistochemistry (FVIII-RAg, IgM, IgG, C3, CSb-9.
`h-CDSS). standard PAS staining and transmission electron microscopy (TEM).
`Results: The median survival time in this cohort of 4 animals was 24.5 days,
`longest survival achieved was 68 days. In all biopsies investigated. despite seemingly
`stable graft function a progressive graft damage was observed. predominantly on
`the glomerular level. TEM analysis revealed profound glomerular basement
`membrane damage over time. On immunohistochemistry this damage was paralleled
`by an increased deposition of recipient IgM immunoglobulins associated with
`subsequent deposition of terminal complement complexes. Discussion: In this pig
`to primate kidney transplantation model. despite profound postoperative CyP
`based immunosuppression and seemingly stable graft function for > two weeks, a
`subacute graft damage likely to be mediated by intragraft recipient antiporcine
`antibody deposition is induced by the recipients humoral immune system.
`
`Poster Board #-Session: P240-I1
`Abstract# 853
`NATURAL REGULATION OFGALUl-3CAL EXPRESSION. JOO HoTai,'
`Jeffrey L. Platt.'.','
`'Surgery. Mayo Clinic, Rochester, MN: 'Immunology.
`Mayo Clinic. Rochester. MN; 'Pediatrics. Mayo Clinic. Rochester, MN.
`One primary hurdle to clinical xenotransplantation IS expression of a sacchande,
`Galal-3Gal. by lower mammals such as pigs. This saccharide is recognized by
`xenoreactive antibodies which initiate the vascular rejection of pig-to-primate
`xenografts. The expression of Galal-3Gal is presumably controlled by the
`availability of core saccharide precursor, nucleotide sugars and the reciprocal actions
`of a- I .3-galactosyltransferase and a-galactosidase. To elucidate the mechanisms
`controlling expression of Galal-3Gal. we studied porcine testis. a tissue which
`exhibits a dramatic transition of Galal -3Gal expression. using lectins, and antibodies
`
`directed against a-I .3-galaciosyltr~~~erase and a-galactosidase. To localize porcine
`a-galactosidase as well as porcine a- I ,3-galactosyltransferase. we cloned the
`corrcsponding genes by 5'/3' RACE (rapid amplification of cDNA ends) and
`developed antibodies against deduced amino acid sequences. Primary spermatocytes,
`but not more mature spermatocytes bound the 1B4 lectin. indicating expression of
`Galal -3Gal. Spermatogonia bound UEA-I and secondary spermatocytes and some
`of spermatids bound LEL and RCA-I lectins instead of 184. suggesting loss of
`terminal a-galactosyl residues or de noro synthesis of core structure with maturation
`toward the center of seminiferous tubules. Munne monoclonal antibodies specific
`for porcine bound to senoli cells and spermatozoa in spcrmiation. apparently in
`middle pieces of tail. while monoclonal antibodies specific for porcine a-galactosidase
`bound 10 sertoli cells and the region including secondary spermatocytes, spermatids
`and residual bodies. These results suggest that the dramatic transition of Galal-
`3Gal expression in seminiferous tubules might be caused by the activity of a-
`galactosidase and/or possibly by the cleavage of acceptor for a-1.3-
`galactosyltrdnsfere. This observation offers a potential means by which Galal -
`3Gal expression might be modulated.
`
`349
`
`

`

`ANTIGEN PRESENTATION
`
`Poster Board #-Session: P241-I1
`Abstract# 854
`PERIPHERAL SURVIVAL OF MURINE C M T CELLSSELECTED IN
`PORCINE THYMUS CRAIWS. Jose-Ignacio Rdriguez-Barbosa,l Yong
`Zhao,' Gui-Ling Zhao,' Sheng-Ping Wang,' David H. Sachs,' Megan Sykes.'
`'Bone Marrow Transplantation Section. Transplantation Biology
`Research Center, Massachusetts General HospitallHarvard Medical
`School, Boston, MA.
`Background: We have demonstrated that thymus transplantation IS capable of
`inducing tolerance across xenogeneic barriers. Studies in T cell receptor transgenic
`mice showed that positive selection in porcine thymus grafts is mediated entirely
`by porcine MHC. Some reports suggest that T cells positively selected on a
`piuticular thymic MHC require the same MHC in the penphery to survive. This
`observation may pose a problem for the strategy of using xenogeneic thymic
`transplantation for the induction of tolerance. since T cells are selected on an MHC
`different from that they will see in the periphery. Methods: We compared the
`decay of mouse CD4 T cells selected in fetal porcine thymus (FPTHY) grafts with
`that of T cells selected in syngeneic neonatal mouse thymus (NMTHY) grafts
`following removal of
`the thymic graft from thymectomized (ATX) conditioned
`class 11 wild type (WT) and class 11 knock-out (KO) mice. Munne CD4 repopulation
`was followed by FACS. These studies assessed whether continual CD4 cell
`repopulation in conditioned FPTHY WT mice requires continual output of T cellc
`from porcine thymic grafts to compensate for rapid death of T cells . Results :
`Conditioned ATX class 11 WT and class II KO mice grafted with either FPTHY
`or syngeneic NMTHY reconstituted the penpheral CD4 T cell compartment to the
`same extent. Following graftectomy at 13 weeks. a gradual decline was observed
`among CD4' PBL and naive-type CD4 cells in class 11 WT and class II KO mice
`grafted with NMTHY. Only at 15 weeks after Gra!Tx was it possible to detect a
`statistically significant increase in the decay of CD4 cells in GrafTx class I1 KO
`compared to WT controls (p4.05). The decline of the % C W PBL and naive-
`type CD4 (CD4+CD44"w) cells after GrafTx was also gradual and similar in class
`11 WT mice grafted with either NMTHY or FFTHY. Conelusion : A munne CD4
`T cell repertoire positively selected on porcine thymus is able to survive in the
`periphery of mice, despite the apparent absence of porcine antigen presenting
`cells. The same class I1 in the periphery as that of the thymus in which T cells were
`selected may not play a cntical role in the survival of T cells selected on a highly
`disparate xenogeneic MHC.
`
`Abstract# 855
`Poster Board #-Session: P242-I1
`ROLE OF PIG CYTOKINES IN INDUCTION OF XENOGENEIC BONE
`MARROW CHIMERISM IN A DISCORDAWPIGTO MOUSE MODEL
`Muhammad M. Mohiuddin,! Yuru Meng,' Verdi J. DiSesa.' 'Cardiovascular-
`Thoracic Surgery, Rush Presbyterian St Luke's Medical Center, Chicago,
`IL.
`Purpose :Xenografts from pigs might solve the problem of the shortage of donor
`organs. The immune mechanisms of xenograft rejection are complex and cannot be
`controlled by conventional immunosuppressive drugs. Induction of donor specific
`tolerance would over come this bamer. Mixed xenogeneic chimerism might be an
`effective method of inducing tolerance to xenografts. In this study we demonstrate
`the importance of pig cytokines in long term survival of pig bone marrow cellc in a-
`Gal knockout (KO) and CS7BL6 (WT) mice. Methods : Pig hone marrow was
`extracted from nbs and a single cell suspension of whole bone marrow ( 60 -100 X
`100celIs) was injected into sub-lethally irradiated (6SOcCy) a-Gal KO (Group A)
`and WT (Group B) mice(n=lO in each group). Both groups of mice received 0.1 ml
`of Anti Lymphocyte Serum (ALS) on day -1 and a single dose(SOmg/Kg) of
`Cyclophosphamide (CyP) on day 2. Half of the mice from each group received Pig
`IL3 and GM-CSF (IOpgKg) daily. All mice were typed by flow cytometry every
`4 weeks using Lectin-FITC and SLA-DR antibodies. Results: At four weeks Group
`A mice which received cytokines showed significani multi-lineage engraftment of
`pig cells (6.8%) whereas, mice not receiving cytokines \howed no engraftment of
`pig cells (0%). In group B mice which received cytokines, a very small number of
`pig cells were detected (2.3%) whereas, in mice whichdid not received any cytokine\.
`a high number of pig cells was detected (12-14%). At eight week\ only group A
`mice receiving cytokines and group B mice not receiving cytokines had permtcnce
`of multilineage pig cells (2-6%). No pig cells were dctccted at eight weeks in the
`other two groups. Conclusion: We have demonstrated that stable engraftment of
`pig cells in KO mice is dependent on pig cytokines whereas in wild type mice pig
`cytokines are not required. Perhaps, in KO mice cytokine producing cells in the pig
`bone marrow inoculum are suppressed or killed by the anti a-Gal antibodies whereas.
`in WT mice this bamer is absent. This study also indicates that the WT mou\e
`hematopoietic environment is sufficient for the growth of pig bone marrow cells.
`
`Abstract# 856
`Poster Board #-Session: P243-I1
`ACllVA"IONOFHUMANDENDRITICCEL,LSBY PORCINEAORTIC
`ENDOTHELIAL CELLS: TRANSACTIVATION OF NAIVE T CELLS
`THROUGH C(ISIIMULATIONANDCYTOKINEGENERATI0N. Partha
`P. Manna, T. Mohanakumar.
`Background: Debdritic cells (DCs) are mosi potent antigen presenting cells in the
`immune system. In order 10 define the role of human DC in human anti-porcine
`immune responses. we analyzed the interaction of human DC with porcine aortic
`endothelial cells(PAEC).
`Methods, To determine the immune responses, both monocyte derived and
`peripheral blood D C s was cultured with porcine and human endothelial cells. We
`analyzed the role of CDI la, CDI I band CDS4 in cell to cell adhesion assay using
`antibodies against these molecules. The expression pattern of co-stimulatory
`molecules (CD40,CDXO,CD86),adhesion molecule (CDS4), and intracellular
`cytokines (IL-12~70 and TNF-U) in DC following interaction with endothelial
`cells was determined by immunofluorescence.
`Results: Human DC adhered to PAEC (3R-402) and this adhesion was augmented
`(>SO%) upon treatment with either recombinant swine interferon-y or recombinant
`human TNF-a. Addition of human DC to PAEC was blocked by pretreatment of
`DC with antibodies specific to human LFA-I and CDS4. Adhesion of DC to PAEC
`also resulted in the activation of DC which was manifested by upregulation of co-
`stimulatory molecules (CLMO,CDXO,CDX6), adhesion molecule (CD54) and HLA-
`DR. PAEC activated human DC provided proliferative signals to the naive autologous
`CD4+ T cells and synthesized IL-12~70 and TNF-a. However, activated DCs
`failed to lyse PAEC in such interaction.
`Conclusion: Human DCs effectively adhered to PAEC and are activated by
`xenoantigen resulting in highly efficient antigen presentation and proliferation of
`CD4+ T cells. Further, this interaction of human DC to PAEC is regulated by the
`participation of co-stimulaiory molecules (CD40.CDRO.CD86). adhesion molecules
`(LFA-I.CDS4) and cytokines (IL-12p70,TNF-a).
`
`CONCURRENT SESSION 41 :
`h G E N PRESENTATION
`
`Abstract# 857
`Uh"PRIMEDCD&TCELLSAREACI'IVATEDBY RESIINGVASCULAR
`ENDOTHELIUM TO INDUCE ENDOTHELIAL CELL APOPTOSIS.
`Alexander S. Krupnick,l Daniel Kreisel.' Wilson Y. Szeto,' Sicco H. P0pma.l
`Bruce R. Rosengard.' 'Department nf Surgery. Hospital of the University of
`Pennsylvania, Philadelphia, PA.
`Purpose: Immunologic destruction of endothelium contributes to acute rejection as
`well as chronic allograft vasculopathy. Although activated vascular endothelium
`has been classified as a semiprofessional antigen presenting cell, it IS controversial
`whether resting vascular endothelium present at the blood/allograft interface can
`activate allogeneic T cells without help from profesbional antigen presenting cells
`(APC). Our purpose is to define this interaction in an in vitro system.
`Methods: Vascular endothelial cells from the thoracic aorta of CS7BW6 (H-2Kh)
`mice were isolated and cultured free of professional APC using a recently developed
`method. CD8+ T lymphocytes were punfied from splenocytes and pooled lymph
`node cells of TCR transgenic (reactive against H-2Kh) BM3(H2KL) mice using
`nylon wool depletion of adherent cells and positive selection with immunomagnetic
`beads. The CDX+ T cells were labeled with CFSE and proliferation was tracked
`using flow cytometry on day\ 2.4. and 6 Apoptosis of the endothelium was
`evaluated concurrently by TUNEL staining.
`Rewlts. Only minimal T cell proliferation ((1.5% and 3%)was seen on days 2 and 4
`respectively. but SS% of the T cells had proliferated by day 6. Accordingly only
`0.5% and 16% of the endoihelial targets were destroyed by ihe alloreactive T cells
`on days 2 and 4. while 41% of the endothelium was undergoing apopto\is 6 days
`after co-culture compared to control\.
`Conclusion: We have demonstrated that mouse vascular endothelium can activate
`unpnmed alloreactive CDX+ T lymphocytes which then induce endothelial cell
`apoptosis. These results sugge\t that resting vascular endothelium can act as an
`antigen presenting cell for CDX+ direct allorecognition and may therefore play an
`important role in regulating immune processes of acute and chronic allograft rejection.
`Since endothelium remains at the blWallograft interface for the life of the patient,
`these finding have important immunological consequences for the field of organ
`transplantation.
`
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`

`

`Abstract# 858
`INTERACTIONS AMONG RECIPIENT MONOCYTES AND DONOR
`I N T H E M A I " A N C E 0 F T H E I P A T H W A Y
`OF ALLORECOGNITION. Dmitry V. Samsonov,' Christopher S. Geehan,'
`Mark D. Denton,' Gilles Benichou,2 Ana Maria Waaga,l Mohamed H.
`Sayegh,' David M. Briscoe.' 'Division ofNephrology. Children's Hospital,
`Boston, MA; ZSchepens Eye Research Institute, Harvard Medical School,
`Boston, MA.
`Endothelial cells (EC) promote the differentiation of monocytes into immature
`APCs. These immature APCs are primed to phagocytose apoptotic and necrotic
`cells within the graft. We have shown that monocyte-endothelial interactions that
`promote APC generation also induce the production of proinflammatory cytokines
`by the APC. We now propose that this function of EC facilitates the maintenance
`of Tcell activation via the indirect pathway of allorecognition. To test this possibility,
`we first cultured PBMCs with allogeneic EC for 6 days. CD14+ monocytes were
`isolated from the coculture and were used as stimulators with autologous CD4+ T
`cells. In these studies, the proliferation of T cells was 3-6 times higher in the
`presence of these EC-modified monocytes as compared to unmodified monocytes
`cultured on plastic. Thus, EC either enhance the costimulatory capacity of APCs.
`or/and promote proliferation via the indirect pathway of allorecognition. To assess
`the latter possibility, we compared proliferation of CD4+ T cells to the direct or the
`indirect pathway when EC were used as a source of alloantigen. CD4+ T cells were
`cultured with irradiated self-APCs in the presence of either allogeneic EC monolayers
`or sonicated EC. T cell proliferati